Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification

Abstract

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.

FIG. 1.

Oligonucleotide primers used for amplification of tcdB. Single-underlined and double-underlined letters indicate the sequences of primers for LAMP and for nested PCR, respectively.

FIG. 2.

Real-time detection of turbidity (A) and 5% polyacrylamide gel electrophoresis (B) of amplification products by LAMP. The template extracted from strain VPI 10463 in 10-fold serial dilutions from 50 ng to 50 ag per reaction tube (lanes 1 to 10) was added. Lane 11, negative control; lanes M, 100-bp ladder as a molecular size marker.